The overall goal of this project is to develop new probes of enhanced functionality and stability for in vivo EPR spectroscopy and imaging. Functionally oriented classes of soluble paramagnetic probes such as pH, SH- and NO-sensitive nitroxyl radicals (NR) have been developed but they often suffer from insufficient stability in living tissues. In turn, triarylmethyl (TAM) radicals have been the popular choice for EPR imaging applications offering advantages over nitroxides, that is, stability in cells and tissues, and narrow linewidth, resulting in high analytical resolution at 5M concentrations and enhanced sensitivity to O2. However, until now applications of TAM radicals have been functionally limited, mostly to EPR oximetry. In this project we propose to enhance the functionality of TAM radicals by developing pH sensitive derivatives in addition to oxygen sensing. Taking into account that both oxygen and pH play key roles in cellular metabolism and homeostasis, these probes may become useful tools in biomedical research. The specific aims are: (SA1) to synthesize pH-sensitive trityl radicals. The synthetic route for the incorporation of specific ionizable groups, which provide pH sensitivity to TAM derivatives in the desirable pH range, is proposed. The probes synthesized under the SA1 are the basis for the entire project. (SA2) To define the spectroscopic and physico-chemical characteristics of pH- and O2-sensitive TAMs. Quantitative characterization of the newly synthesized TAMs is absolutely crucial, both for the optimization of the synthetic procedure (i.e. the choice of the most effective probes) and for the efficiency of their further spectroscopic and imaging applications. (SA3) To study the role of myocardial acidosis and oxygen depletion in ischemic hearts and in the model of ischemic preconditioning using newly developed TAMs. The measurement of pH and oxygen depletion in ischemic heart is of principal importance for understanding related biochemical processes believed to contribute to myocardial injury, and will be measured in controlled and preconditioned rat hearts using developed extremely stable probes of dual functionality. The results will contribute to the understanding of preconditioning mechanisms and provide opportunities for designing corresponding therapeutic approaches. In summary, the success of this project may have a significant impact on the future of in vivo EPR spectroscopy and bioimaging applications to medicine.